U.S. patent number 8,394,776 [Application Number 12/523,314] was granted by the patent office on 2013-03-12 for use of ursolic acid saponin,oleanolic acid saponin in preparation of increasing leucocytes and/or platelet medicine.
This patent grant is currently assigned to Chengdu Di'Ao Jiuhong Pharmaceutical Factor. The grantee listed for this patent is Guangxin Dong, Xiaoxia Gong, Bing Guang, Yu Huang, Zhen Huang, Jianxin Ji, Bogang Li, Junjian Liu, Xiangyang Peng, Dongguang Qin, Jufang Yan, Wei Zhan, Meirong Zhou. Invention is credited to Guangxin Dong, Xiaoxia Gong, Bing Guang, Yu Huang, Zhen Huang, Jianxin Ji, Bogang Li, Junjian Liu, Xiangyang Peng, Dongguang Qin, Jufang Yan, Wei Zhan, Meirong Zhou.
United States Patent |
8,394,776 |
Guang , et al. |
March 12, 2013 |
Use of ursolic acid saponin,oleanolic acid saponin in preparation
of increasing leucocytes and/or platelet medicine
Abstract
The invention provides the use of ursolic acid saponin and
oleanolic acid saponin of formula (I) in preparing medicaments for
increasing leucocytes and/or platelets. The invention also provides
a pharmaceutical composition containing the same compound. The
invention utilizes the cheap and accessible ursolic acid and
oleanolic acid which are widely present in natural plants as raw
materials, introduces monosaccharyls or oligosaccharyls by
structural modification. It is proved by pharmacological tests that
the compound of formula (I) have an activity of obviously
increasing leucocytes and/or platelets. ##STR00001##
Inventors: |
Guang; Bing (Sichuan,
CN), Liu; Junjian (Sichuan, CN), Dong;
Guangxin (Sichuan, CN), Peng; Xiangyang (Sichuan,
CN), Gong; Xiaoxia (Sichuan, CN), Huang;
Zhen (Sichuan, CN), Zhou; Meirong (Sichuan,
CN), Zhan; Wei (Sichuan, CN), Yan;
Jufang (Sichuan, CN), Huang; Yu (Sichuan,
CN), Qin; Dongguang (Sichuan, CN), Ji;
Jianxin (Sichuan, CN), Li; Bogang (Sichuan,
CN) |
Applicant: |
Name |
City |
State |
Country |
Type |
Guang; Bing
Liu; Junjian
Dong; Guangxin
Peng; Xiangyang
Gong; Xiaoxia
Huang; Zhen
Zhou; Meirong
Zhan; Wei
Yan; Jufang
Huang; Yu
Qin; Dongguang
Ji; Jianxin
Li; Bogang |
Sichuan
Sichuan
Sichuan
Sichuan
Sichuan
Sichuan
Sichuan
Sichuan
Sichuan
Sichuan
Sichuan
Sichuan
Sichuan |
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A |
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN |
|
|
Assignee: |
Chengdu Di'Ao Jiuhong
Pharmaceutical Factor (Sichuan, CN)
|
Family
ID: |
39635662 |
Appl.
No.: |
12/523,314 |
Filed: |
January 16, 2008 |
PCT
Filed: |
January 16, 2008 |
PCT No.: |
PCT/CN2008/000107 |
371(c)(1),(2),(4) Date: |
March 26, 2010 |
PCT
Pub. No.: |
WO2008/086739 |
PCT
Pub. Date: |
July 24, 2008 |
Prior Publication Data
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|
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Document
Identifier |
Publication Date |
|
US 20100197898 A1 |
Aug 5, 2010 |
|
Foreign Application Priority Data
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|
|
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Jan 16, 2007 [CN] |
|
|
2007 1 0048277 |
Jan 16, 2007 [CN] |
|
|
2007 1 0048278 |
|
Current U.S.
Class: |
514/33 |
Current CPC
Class: |
A61K
31/704 (20130101); A61P 7/00 (20180101) |
Current International
Class: |
A01N
43/04 (20060101); A61K 31/70 (20060101) |
Foreign Patent Documents
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1210864 |
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Mar 1999 |
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CN |
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1355172 |
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Jun 2002 |
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CN |
|
1446818 |
|
Oct 2003 |
|
CN |
|
1472220 |
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Feb 2004 |
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CN |
|
1593436 |
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Mar 2005 |
|
CN |
|
1628679 |
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Jun 2005 |
|
CN |
|
1682748 |
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Oct 2005 |
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CN |
|
1788758 |
|
Jun 2006 |
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CN |
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2000-256391 |
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Sep 2000 |
|
JP |
|
2006-206468 |
|
Aug 2006 |
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JP |
|
2006/112932 |
|
Nov 2006 |
|
KR |
|
03/045410 |
|
Jun 2003 |
|
WO |
|
Other References
Vippagunta et al. Advanced Drug Delivery Reviews 48 (2001) 3-26.
cited by examiner .
Matsuda et al. Life Sciences, vol. 63.No. 17, pp. PL 245-250, 1998.
cited by examiner .
Gao. CN 1788758 A, Jun. 21, 2006, machine translation. cited by
examiner.
|
Primary Examiner: Bland; Layla
Attorney, Agent or Firm: Occhiuti Rohlicek & Tsao
LLP
Claims
The invention claimed is:
1. A method for increasing the number of leucocytes or platelets in
a subject in need thereof, the method comprising administering to
the subject an effective amount of 3-O-(.alpha.-L-arabopyranosyl)
ursolic acid-28-O-(.alpha.-L-arabopyranosyl) ester.
2. A method for increasing the number of leucocytes or platelets in
a subject in need thereof, the method comprising administering to
the subject an effective amount of 3-O-(.alpha.-L-arabopyranosyl)
oleanolic acid-28-O-(.alpha.-L-arabopyranosyl) ester or
3-O-(.beta.-D-glucopyranosyl) oleanolic
acid-28-O-(.beta.-D-glucopyranosyl) ester.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the National Stage of International Application
No. PCT/CN2008/000107, filed on Jan. 16, 2008, which claims the
benefit of Chinese Application Serial No. 200710048277.1, filed on
Jan. 16, 2007 and Chinese Application Serial No. 200710048278.6,
filed on Jan. 16, 2007, the contents of the foregoing applications
are hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION
The present invention falls within the field of pharmaceutical
technology. Particularly, it relates to the use of ursolic acid
saponin and oleanolic acid saponin in the preparation of
medicaments for increasing leucocytes and/or platelets.
BACKGROUND OF THE INVENTION
Due to the phagocytosis of leucocytes, they can eliminate foreign
pathogens and safeguard the health of the human body. So, they are
called the body defender. There are 4,000-10,000 leucocytes per
mm.sup.3 in the system of blood circulation of a normal human body.
The most important leucocytes are neutrophilic granulocytes having
a defensive function, accounting for 50-70% of the total count.
Generally, the most frequent and most common leukopenia is
neutropenia. When the total leucocyte count is less than
4,000/mm.sup.3, body resistance is so low that bacterial infection
is easily caused and life is threatened in severe cases. Platelets
are one of the blood visible components. The number of platelets in
human blood varies greatly, with a normal value of
150,000-300,000/microlitre. Platelets have the functions of
stopping bleeding and engulfing viruses, bacteria and other
particles, and can nourish and support the capillary endothelial
cells to decrease capillary fragility. If the number of platelets
significantly decreases or some function disorder occurs, it will
lead to a bleeding tendency. Leukopenia and/or thrombocytopenia is
common in clinical, including a primary type, a concurrent type and
a secondary type. In addition to geneogenous leukopenia and/or
thrombocytopenia, there are a variety of causes. Diseases, drugs,
radiations, infections, toxins, chemicals, surgical treatments,
environmental factors and the like are all likely to cause
leukopenia and/or thrombocytopenia. The diseases causing leukopenia
and/or thrombocytopenia include hematopoietic system diseases,
immune system diseases, infections, systemic lupus erythematosus,
allergic shock and thrombocytopenic purpura, etc. Both
myeloproliferative disorder and aplastic anemia may be accompanied
with leukopenia and/or thrombocytopenia. Bone marrow transplant and
liver transplant also directly result in the decrease of leucocytes
and/or platelets. Especially for cancer patients, radiotherapy and
chemotherapy often cause inhibition of bone marrow hematopoiesis,
of which the manifestation is an obvious decrease of peripheral
leucocytes and platelets. At present, drugs for treating various
types of leukopenia and/or thrombocytopenia are not many yet. The
effect of products of genetic engineering, such as granulocyte
colony-stimulating factor (GCSF) and granulocyte-macrophage
colony-stimulating factor (GMCSF), in increasing leucocyte is
remarkable. The Chinese traditional medicine, Sanguisorba Tablet is
widely used to increase the number of Leucocytes too, while there
are few chemical medicines with significant therapeutic efficacy.
Thus, there is a wide need of chemical medicines which can be
effective in treating leukopenia and/or thrombocytopenia,
convenient to use, easy to control the quality, and easy to obtain
by synthesis.
Pentacyclic triterpenes, one type of the naturally distributed
triterpenes compounds, are found to have a wide physiological
activity. Ursolic acid and oleanolic acid, the most representative
compounds thereof, are widely present in the plant kingdom and can
be obtained in a large amount. Oleanolic acid has been used for the
treatment of liver diseases for many years. In addition to the
beneficial effect on liver, both have a variety of other activities
according to reports. In the article entitled "Effects of oleanolic
acid and ursolic acid on inhibiting tumor growth and enhancing the
recovery of hematopoietic system postirradiation in mice (Cancer
letter 7-13, 111, 1997)", Lin et al. studied the effect of ursolic
acid and oleanolic acid in inhibiting tumor cell growth and found
that ursolic acid and oleanolic acid were effective in increasing
leucocytes when they were administrated through abdominal cavity in
the doses of 25, 50 and 100 mg/kg, and the effect was obvious in
the case of a high dose, wherein ursolic acid was more effective
than oleanolic acid. Japanese patent No. JP7048260 also discloses
the effect of ursolic acid in increasing erythrocytes and
platelets. Chinese patent No. CN03135776 discloses the remarkable
effect of triterpenoid saponins in increasing leucocytes and
platelets, wherein the triterpenoid saponins are isolated from
sanguisorba and mainly have 19-hydroxy ursolic acid (also known as
pomolic acid) as an aglycon. Since sanguisorba comprises a series
of saponins of such type with similar structures and polarities and
with a limited content, it is difficult to separate out and purify
the effective monomers in batches for medical use; in addition, the
pomolic acid or the aglycon thereof per se is not widely present in
natural products, thus it is not easy to obtain them in a large
amount in the sight of isolation and extraction; and in the sight
of synthesis, it is not easy to obtain 19-hydroxy via simple
conversion procedures original from ursolic acid, as a raw
material, which is easy to obtain, thus there are certain
limitations of its exploitation and utilization.
So far, the use of ursolic acid saponins and oleanolic acid
saponins in the preparation of medicaments for increasing
leucocytes and/or platelets has not been reported. In the present
invention, ursolic acid and oleanolic acid, which are widely
distributed in natural products, cheap and easy to get, being
creatively used as raw materials, and with the introduction of
hydrophilic groups, i.e. monosaccharyls or oligosaccharyls by
structural modification, it has been proved that saponins can not
only improve the water solubility of the parent nucleus, but also
have an activity of remarkably increasing leucocytes and/or
platelets by pharmacological tests. Compared to pomolic acid
saponin isolated from sanguisorba, the patented compounds not only
show up stronger pharmacological activities, but also have
advantages of synthesis of simple steps, adaptation to industrial
production, low cost and so on.
SUMMARY OF THE INVENTION
The technical solution of the present invention provides the use of
ursolic acid saponins and oleanolic acid saponins of formula (I) or
their pharmaceutically acceptable salts and esters in preparing
pharmaceuticals for increasing leucocytes and/or platelets. Formula
(I) is:
##STR00002##
wherein R1 is hydrogen, glucosyl, arabinosyl, rhamnosyl,
galactosyl, xylosyl, ribosyl, lyxosyl, mannosyl, allosyl, altrosyl,
gulosyl, fructosyl, sorbosyl, quinovosyl, fucosyl, piscosyl,
2-aminoglucosyl, galacturonyl, glucuronyl, or oligosaccharyl formed
of 2-5 of such monosaccharide;
R2 is hydrogen, glucosyl, arabinosyl, rhamnosyl, galactosyl,
xylosyl, ribosyl, lyxosyl, mannosyl, allosyl, altrosyl, gulosyl,
fructosyl, sorbosyl, quinovosyl, fucosyl, piscosyl,
2-aminoglucosyl, galacturonyl, glucuronyl, or oligosaccharyl formed
of 2-5 of such monosaccharides; and
R1 and R2 are not hydrogen at the same time.
The compound is ursolic acid saponin when R3 is CH.sub.3 and R4 is
H, represented by formula (II):
##STR00003##
Further, when G1 and G2 of formula (II) are the same monosaccharyl
or oligosaccharyl, it is called bisglycosyl ursolic acid saponin.
When G1 and G2 are hydrogen, monosaccharyl or oligosaccharyl and G1
is different from G2, it is called non-bisglycosyl ursolic acid
saponin.
Wherein, said glycosyl is glucosyl, arabinosyl, rhamnosyl,
galactosyl, xylosyl, glucuronyl, or oligosaccharyl formed of 2-5 of
such monosaccharides.
Further, bisglycosyl ursolic acid saponin of formula (II) is
selected from the group consisting of:
3-O-(.alpha.-L-arabopyranosyl) ursolic
acid-28-O-(.alpha.-L-arabopyranosyl) ester,
3-O-(.alpha.-L-rhamnopyranosyl) ursolic
acid-28-O-(.alpha.-L-rhamnopyranosyl) ester, and
3-O-(.beta.-D-glucopyranosyl) ursolic
acid-28-O-(.beta.-D-glucopyranosyl) ester.
Further, non-bisglycosyl ursolic acid saponin of formula (II) is
selected from the group consisting of:
3-O-(.beta.-D-galactopyranosyl) ursolic acid,
3-O-(.alpha.-L-rhamnopyranosyl) ursolic acid,
3-O-(.alpha.-L-arabopyranosyl) ursolic acid,
3-O-(.beta.-D-glucopyranosyl) ursolic acid,
3-O-(.alpha.-L-arabopyranosyl) ursolic
acid-28-O-(.beta.-D-glucopyranosyl) ester,
3-O-(.alpha.-L-arabopyranosyl) ursolic
acid-28-O-(.beta.-cellobiosyl) ester, and
ursolic acid-28-O-(.alpha.-L-arabopyranosyl) ester.
Still further, bisglycosyl ursolic acid saponin of formula (II) is
preferably selected from the group consisting of:
3-O-(.alpha.-L-arabopyranosyl) ursolic
acid-28-O-(.alpha.-L-arabopyranosyl) ester,
3-O-(.alpha.-L-arabopyranosyl) ursolic acid and its sodium
salts,
3-O-(.alpha.-L-arabopyranosyl) ursolic
acid-28-O-(.beta.-D-glucopyranosyl) ester, and
3-O-(.beta.-D-glucopyranosyl) ursolic
acid-28-O-(.beta.-D-glucopyranosyl) ester.
Still further, the compound of formula (II) is more preferably
selected from:
3-O-(.alpha.-L-arabopyranosyl) ursolic
acid-28-O-(.alpha.-L-arabopyranosyl) ester.
The present invention also provides the use of ursolic acid
saponins of the above formula (II) in preparing a pharmaceutical
for increasing leucocytes and platelets.
According to the use of ursolic acid saponin and oleanolic acid of
formula (I), when R3 is H and R4 is CH.sub.3, the compound is
oleanolic acid saponin, represented by formula (III):
##STR00004##
Wherein, G3 and G4 are hydrogen, glucosyl, arabinosyl, rhamnosyl,
galactosyl, xylosyl, glucuronyl, or oligosaccharyl formed of 2-5 of
such monosaccharides, and G3 and G4 are not hydrogen at the same
time.
Further, the compound of general formula (III) is selected from the
group consisting of:
3-O-(.beta.-D-glucuronopyranosyl) oleanolic acid,
3-O-(.beta.-D-xylopyranosyl) oleanolic acid,
3-O-(.alpha.-L-galactopyranosyl) oleanolic acid,
3-O-(.alpha.-L-arabopyranosyl) oleanolic acid and its sodium
salts,
3-O-(.beta.-D-glucopyranosyl) oleanolic acid and its sodium
salts,
3-O-(.alpha.-L-arabopyranosyl) oleanolic
acid-28-O-(.alpha.-L-arabopyranosyl) ester,
3-O-(.beta.-D-glucopyranosyl) oleanolic
acid-28-O-(.beta.-D-glucopyranosyl) ester,
oleanolic acid-28-O-(.beta.-gentiobiosyl) ester,
oleanolic acid-28-O-(.beta.-D-glucopyranosyl) ester.
Still further, the compound of formula (III) is selected from the
group consisting of:
3-O-(.alpha.-L-arabopyranosyl) oleanolic
acid-28-O-(.alpha.-L-arabopyranosyl) ester, and
3-O-(.beta.-D-glucopyranosyl) oleanolic
acid-28-O-(.beta.-D-glucopyranosyl) ester.
The present invention also provides the use of oleanolic acid
saponins of the above formula (III) in preparing pharmaceuticals
for increasing platelets.
Wherein, said pharmaceuticals for increasing leucocytes and/or
platelets are used to prevent and treat primary, concurrent and
secondary leukopenia and/or thrombocytopenia in mammals.
The said pharmaceuticals for increasing leucocytes and/or platelets
can also be used separately or in combination to prevent and treat
infectious diseases.
Wherein, said leukopenia and/or thrombocytopenia may be caused by
diseases, drugs, radiations, infections, toxins, chemical
substances, surgical treatments and environmental factors.
Wherein, diseases that cause leukopenia and/or thrombocytopenia
include hematopoietic system diseases, immune system diseases,
infective diseases, systemic lupus erythematosus(SLE), anaphylactic
shock, thrombocytopenic purpura and chemicotherapy for cancer
patients.
The present invention provides a pharmaceutical composition useful
for increasing human leucocytes and/or platelets, which is a
pharmaceutical preparation prepared from any one of the compounds
of formulae (I), (II) and (III) or their pharmaceutically
acceptable salts, hydrates or solvates of the salts, esters or
prodrugs, as an active ingredient, in combination with
pharmaceutically acceptable excipients or carriers.
Further, said pharmaceutical preparations may be in the form of
tablets, capsules, pills, injections, sustained-release
preparations, controlled-release preparations, or various
microparticulate drug delivery systems.
According to the general rules of body metabolism and the common
knowledge comprehensible to those skilled in the art of
pharmacochemistry, the compounds of formulae I-III can be derived
by ester linkage, and those compounds of formulae I-III with acidic
or alkaline radicals can also be prepared into pharmaceutically
acceptable salts. The use of such pharmaceutically acceptable
salts, hydrates or solvates of the salts, or esters, particularly
predrugs which can be metabolized in vivo into the compounds of
formulae I-III, in preparing pharmaceuticals for increasing
leucocytes and/or platelets is also covered by this invention.
Also, the use of the traditional Chinese medicine, herbal medicine
and their effective parts containing any one of the compounds of
formulae I-III in preparing pharmaceutical for increasing
leucocytes and/or platelets is covered by this invention. Still
further, this invention also covers the use of a pharmaceutical
composition in preparing medicaments for increasing leucocytes
and/or platelets, the pharmaceutical composition comprising any one
of the compounds of formulae I-III and their pharmaceutically
acceptable salts, hydrates or solvates of the salts, esters and
prodrugs, the traditional Chinese medicine, herbal medicine and
their effective parts containing any one of the compounds of
formulae I-III, and pharmaceutically acceptable carriers. The
pharmaceutical composition may be in the form of tablets, capsules,
pills, injections, sustained-release preparations,
controlled-release preparations, or various microparticulate drug
delivery systems, and its use in preparing pharmaceuticals for
increasing leucocytes and/or platelets is covered by this
invention, too.
DETAILED DESCRIPTION OF THE INVENTION
The following examples are used to illustrate this invention in
detail but should not be construed as limiting the scope of the
invention. The synthetic raw materials used in the invention, such
as ursolic acid and oleanolic acid, are commercially available.
Example 1
Preparation of 3-O-(.alpha.-L-arabopyranosyl) ursolic
acid-28-O-(.alpha.-L-arabopyranosyl) ester
1 g (2.2 mmol) of ursolic acid was mixed with 3.4 g (5.7 mmol, 2.6
eq) of
2,3,4-tri-O-benzoyl-.beta.-L-arabopyranosyl-trichloroacetimidate
(See J. Org. Chem. 1999, 64, 7265-7266 for the synthesis method of
the compound) as an arabinose glycosyl donor and 1.5 g of powdered
4 .ANG. molecular sieve in 20 ml of a dry CH.sub.2Cl.sub.2 solution
under N.sub.2 atmosphere, then 0.03 ml (0.15 eq) of trimethylsilyl
trifluoromethanesulfonate (TMSOTf) was added dropwise at
-10.degree. C. The reaction mixture was raised to room temperature
gradually and stirred overnight, then a small amount of Et.sub.3N
(0.3 ml) was added to quench the reaction. After filtration and
concentration of the filtrate, the mixture was subjected to silica
gel column chromatography and eluted with petroleum ether/ethyl
acetate (4/1-2/1) system, and 1.47 g (1.1 mmol) of bisglycosyl
intermediate was obtained. The intermediate was dissolved in 30 ml
of methanol/dichloromethane (2/1) and 378 mg of NaOMe (7 mmol, 6.4
eq) was added to react for 4 h at room temperature. The reaction
mixture was neutralized with acid resin DowexH.sup.+, filtered and
concentrated, and subjected to silica gel column chromatography
(chloroform/methanol, 10/1-6/1). Finally, 0.61 g of white powder
was obtained with a total yield of 39%.
[.alpha.].sub.D.sup.20=+29.0 (C0.72, MeOH)
IR(KBr)cm.sup.-1: 3330, 2958, 1724, 1068, 926, 852
.sup.1HNMR (600 MHz, CD.sub.3OD): .delta.ppm 5.40 (d, J=5.8 Hz, 1H,
H-1''), 5.28 (t, 1H, H-12), 4.30 (d, J=6.8 Hz, 1H, H-1'), 3.81-3.90
(m, 4H), 3.67-3.71 (m, 2H), 3.51-3.60 (m, 4H), 3.15 (dd, J=11.8,
4.4 Hz, 1H, H-3), 2.29 (d, J=11.6 Hz, 1H, H-18), 1.13, 1.06, 0.98,
0.86, 0.84 (s each, 3H each, Me.times.5), 0.97 (3H, d, J=4.0 Hz),
0.91 (3H, d, J=6.4 Hz), 0.79 (d, J=11.6 Hz, 1H, H-5)
.sup.13CNMR (150 MHz, CD.sub.3OD): .delta.ppm 176.5 (C-28), 137.9
(C-13), 125.9 (C-12), 105.7 (C-1'), 94.4 (C-1''), 89.3 (C-3), 72.9,
72.2, 71.4, 69.9, 68.1, 67.0, 64.9 (d), 55.6, 52.7, 48.2, 48.0,
41.9, 39.6, 39.0, 38.8 (d), 38.6, 36.5, 36.2, 32.9, 30.3, 27.9,
27.2, 25.7, 23.7, 23.0, 22.6, 20.1, 17.9, 16.5, 16.2, 15.6,
14.7
ESI-MS (m/z): 743.3 [M+Na].sup.+
Example 2
Preparation of 3-O-(.alpha.-L-rhamnopyranosyl) ursolic
acid-28-O-(.alpha.-L-rhamnopyranosyl) ester
Similar to the method in Example
1,2,3,4-tri-benzoyl-.beta.-L-rhamnopyranosyl-trichloroacetimidate
was used as a glycosyl donor, and 3-O-(.alpha.-L-rhamnopyranosyl)
ursolic acid-28-O-(.alpha.-L-rhamnopyranosyl) ester was
obtained.
[.alpha.].sub.D.sup.20=-11.8 (C0.17, DMF)
.sup.1HNMR (600 MHz, d.sub.5-pyridine): .delta.ppm 6.75 (1H, s),
5.46 (1H, m), 5.32 (1H, s), 4.56-4.57 (2H, m), 4.52 (1H, dd,
J=9.12, 3.2 Hz), 4.47 (1H, dd, J=8.9, 3.2 Hz), 4.38 (1H, t, J=9.3
Hz), 4.29-4.34 (3H, m), 3.17 (H, dd, J=11.8, 4.44 Hz), 2.44 (1H, d,
J=11.3 Hz), 1.81 (1H, m), 1.70 (3H, d, J=6 Hz), 1.67 (3H, d, J=6
Hz), 1.17 (3H, s), 1.08 (1H, m), 0.93 (3H, s), 0.92 (6H, s), 0.90
(3H, d, J=6.4 Hz), 0.88 (3H, d, J=6.4 Hz), 0.87 (3H, s), 0.70 (1H,
d, J=11.7 Hz)
.sup.13CNMR (150 MHz, CD.sub.3OD): .delta.ppm 175.6 (C-28), 138.1
(C-13), 126.1 (C-12), 103.0 (C-1'), 93.6 (C-1''), 89.0 (C-3), 72.7,
72.0, 71.2, 71.1 (d), 71.0, 70.0, 68.5, 55.3, 53.1, 48.5, 48.2,
41.9, 40.0, 39.5, 39.0, 38.6, 38.4, 36.7, 36.5, 33.0, 30.2, 27.6,
27.4, 25.2, 23.8, 23.0, 22.7, 20.0, 18.0, 16.8, 16.4, 16.1, 15.6,
14.6
ESI-MS (m/z): 771 [M+Na].sup.30
Example 3
Preparation of 3-O-(.beta.-D-glucopyranosyl) ursolic
acid-28-O-(.beta.-D-glucopyranosyl) ester
Similar to the method in Example
1,2,3,4,6-tetra-O-benzoyl-.alpha.-D-glucosyl-trichloroacetimidate
was used as a glycosyl donor, and 3-O-(.beta.-D-glucopyranosyl)
ursolic acid-28-O-(.beta.-D-glucopyranosyl) ester was obtained.
[.alpha.].sub.D.sup.20=+21.7 (C1.02, MeOH) IR(KBr)cm.sup.-1: 3417,
2925, 1727, 1672, 1456, 1377, 1226, 1075, 1027, 896, 831
.sup.1HNMR (600 MHz, CD.sub.3OD): .delta.ppm 5.33 (d, J=8.3 Hz, 1H,
H-1''), 5.24 (t, 1H, H-12), 4.31 (d, J=7.8 Hz, 1H, H-1'), 3.77-3.84
(m, 2H), 3.65-3.68 (m, 2H), 3.28-3.39 (m, 7H), 3.16-3.18 (m, 2H),
2.22 (d, J=11.2 Hz, 1H, H-18), 1.12, 1.05, 0.96, 0.84, 0.83 (s
each, 3H each, Me.times.5), 0.88 (d, J=6.4 Hz, 3H), 0.78 (d, J=11.6
Hz, 1H, H-5)
.sup.13CNMR (150 MHz, CD.sub.3OD): .delta.ppm 176.5 (C-28), 137.7
(C-13), 125.9 (C-12), 105.3 (C-1'), 94.3 (C-1''), 89.4 (C-3), 77.2,
76.9 (d), 76.3, 74.3, 72.5, 70.3, 69.8, 61.4, 61.1, 55.7, 52.8,
48.7, 48.0, 41.9, 39.6, 39.0, 38.9, 38.8, 38.6, 36.4, 36.1, 32.9,
30.3, 27.9, 27.2, 25.7, 23.9, 23.0, 22.6, 20.1, 17.9, 16.5, 16.2,
15.7, 14.8
ESI-MS (m/z): 803 [M+Na].sup.+
Example 4
Preparation of ursolic acid-28-O-(.alpha.-L-arabopyranosyl)
ester
Ursolic acid (195 mg, 0.43 mmol) and 2,3,4-tri-O--
benzoyl-L-arabopyranosyl-bromide (316 mg, 1.4 eq) were mixed in a
CH.sub.2Cl.sub.2/H.sub.2O (1/1,10 ml) system, K.sub.2CO.sub.3 (151
mg, 2.5 eq) and Bu.sub.4NBr (56 mg, 0.4 eq) were added, then the
mixture was heated under reflux until the reaction was completed.
Then 20 ml of CH.sub.2Cl.sub.2 was added to separate out the
aqueous phase, and the organic phase was washed with water and
brine, dried and spin dried. The residue was directly used for the
next reaction. The above intermediate was dissolved in a
CH.sub.2Cl.sub.2/MeOH (5 ml, 1/1) system and reacted with sodium
methoxide (130 mg, 2.5 mmol) at room temperature overnight. After
being quenched by adding water, the reaction mixture was extracted
with ethyl acetate for several times. The organic phase was
combined, washed with water, dried and spin dried. Then the residue
was subjected to silica gel column chromatography and eluted with
the CHCl.sub.3/MeOH system to obtain 200 mg of white powder with a
yield of 79%.
[.alpha.].sub.D.sup.20=+43.0 (C0.97, MeOH)
.sup.1HNMR (600 MHz, CD.sub.3OD): .delta.ppm 5.38 (1H, d, J=5.8
Hz), 5.26 (1H, t, J=3.6 Hz), 3.85-3.88 (2H, m), 3.64-3.69 (2H, m),
3.54-3.56 (1H, m), 3.14 (1H, dd, J=11.6, 4.5 Hz), 2.26 (1H, d,
J=11.0 Hz), 1.11 (3H, s), 0.97 (3H, s), 0.96 (3H, d, J=5.9 Hz),
0.95 (3H, s), 0.88 (3H, d, J=6.5 Hz), 0.82 (3H, s), 0.77 (3H, s),
0.74 (1H, d, J=11.8 Hz)
.sup.13CNMR (150 MHz, CD.sub.3OD): .delta.ppm 176.5, 137.9, 125.8,
94.4, 78.3, 72.2, 69.9, 67.0, 64.9, 55.4, 52.7, 48.4, 41.9, 39.6,
39.0, 38.8, 38.7, 38.4, 36.7, 36.2, 33.0, 30.3, 29.2, 27.9, 27.4,
26.5, 23.7, 23.0, 22.6, 20.1, 18.1, 16.5, 16.2, 15.0, 14.7
ESI-MS (m/z): 611.0 [M+Na].sup.+
Example 5
Preparation of 3-O-(.alpha.-L-arabopyranosyl) ursolic acid and its
sodium salt
Ursolic acid (1 g, 2.2 mmol), K.sub.2CO.sub.3 (0.6 g) and benzyl
chloride (0.3 ml) were mixed in DMF (10 ml), and heated to
100.degree. C. until the raw materials reacted completely. After
cooling, the reaction mixture was filtered and the filter cake was
washed with DMF. The organic phase was combined, poured into 100 ml
of water, and filtrated to obtain a white crude product, which was
subjected to silica gel column chromatography and eluted with a
petroleum ether/ethyl acetate system to obtain 1.09 g of benzyl
ursolic acid (2.0 mmol). The above dried intermediate, 1.7 g (2.8
mmol, 1.4 eq) of
2,3,4-tri-O-benzoyl-.beta.-L-arabopyranosyl-trichloroacetimidate as
a glycosyl donor, and 1 g of a powdered molecular sieve were mixed
in 15 ml of a dried CH.sub.2Cl.sub.2 under N.sub.2 atmosphere, then
78 .mu.l (0.43 mmol) of trimethylsilyl trifluoromethanesulfonate
(TMSOTf) was added dropwise at -10.degree. C. The raw material of
benzyl ester was tracked by TLC. The reaction was quenched with
Et.sub.3N after it was complete. After the mixture was filtered,
the filtrate was concentrated, subjected to silica gel column
chromatography and eluted with petroleum ether/ethyl acetate system
to obtain 1.65 g (1.67 mmol) of a glycosyl intermediate,
3-O-(2,3,4-tri-O-benzoyl-.alpha.-L-arabopyranosyl)-benzyl ursolic
acid. The intermediate was dissolved in 15 ml of a
methanol/CH.sub.2Cl.sub.2 (1/2), and subjected to hydrogenation at
normal pressure overnight under catalysis of 0.1 g of 5% Pd/C. The
mixture was filtered, then the filtrate was concentrated, subjected
to silica gel column chromatography and eluted with the petroleum
ether/ethyl acetate system to obtain 1.26 g (1.4 mmol) of
3-O-(2,3,4-tri-O-benzoyl-.alpha.-L-arabopyranosyl) ursolic acid.
This product was dissolved in 25 ml of MeOH/CH.sub.2Cl.sub.2 (2/1)
and added with 35 mg (0.65 mmol) of NaOMe to react at room
temperature overnight. Then the reaction solution was neutralized
with a dilute acetic acid solution, concentrated, subjected to
silica gel column chromatography and eluted with a
chloroform/methanol system to obtain 0.737 g (1.25 mmol) of
3-O-(.alpha.-L-arabopyranosyl) ursolic acid with a total yield of
the four-step reaction of 57%.
[.alpha.].sub.D.sup.20=+41.9 (C0.43, DMF)
.sup.1HNMR (600 MHz, CD.sub.3OD): .delta.ppm 5.22 (1H, t, J=3.6 Hz,
H-12), 4.27 (1H, d, J=6.7 Hz, H-1'), 3.82 (1H, dd, J=12.2, 3.4 Hz),
3.79-3.80 (m, 1H), 3.49-3.58 (3H, m), 3.14 (1H, dd, J=11.4, 4.14
Hz, H-3), 2.19 (1H, d, J=11.0 Hz, H-18), 1.11 (3H, s), 1.04 (3H,
s), 0.96 (3H, s), 0.84 (3H, s), 0.84 (3H, s), 0.87 (3H, d, J=6.5
Hz), 0.78 (1H, d, J=11.2 Hz, H-5)
.sup.13CNMR (150 MHz, CD.sub.3OD): .delta.ppm 175.0, 139.6, 126.9,
107.1, 90.7, 74.3, 72.8, 69.5, 66.4, 57.0, 54.4, 48.0, 42.2, 40.8,
40.4, 40.2, 39.9, 38.1, 37.8, 34.3, 31.8, 29.2, 28.6, 27.0, 25.3,
24.4, 24.1, 21.6, 19.3, 17.8, 17.7, 17.0, 16.1
ESI-MS (m/z): 611.0 [M+Na].sup.30
30 mg of 3-O-(.alpha.-L-arabopyranosyl) ursolic acid was dissolved
in methanol/chloroform (4 ml, 3/1), added with 5 mg of NaOMe, then
stirred at room tempreature for 30 min and filtered to separate out
a white solid. The solid was washed with methanol and dried to
obtain 3-O-(.alpha.-L-arabopyranosyl) ursolic acid sodium salt.
Example 6
Preparation of 3-O-(.beta.-D-glucopyranosyl) ursolic acid and its
sodium salt
3-O-(.beta.-D-glucopyranosyl) ursolic acid was obtained through a
four-step reaction with ursolic acid as a starting material and
2,3,4,6-tetra-O-benzoyl-.alpha.-D-glucopyranosyl-trichloroacetimidate
as a glycosyl donor.
[.alpha.].sub.D.sup.20=+27.0 (C0.2, DMF)
.sup.1HNMR (600 MHz, CD.sub.3OD): .delta.ppm 5.22 (t, 1H, H-12),
4.32 (d, J=7.8 Hz, 1H, H-1'), 3.83 (dd, J=11.8, 2.2 Hz, 1H,
H-6'-1), 3.67 (dd, J=12, 5.2 Hz, 1H, H-6'-2), 3.17-3.35 (m, 5H),
2.20 (d, J=11.4, 1H, H-18), 1.11 (3H, s), 1.06 (3H, s), 0.97 (6H, s
Me.times.2), 0.90 (3H, d, J=5.8 Hz), 0.85 (6H, s), 0.78 (d, J=12.4
Hz, 1H, H-5)
.sup.13CNMR (150 MHz, CD.sub.3OD): .delta.ppm 178.9 (C-28), 137.0
(C-13), 126.2 (C-12), 103.8 (C-1'), 88.0 (C-3), 75.4, 74.8, 72.8,
68.8, 59.9, 54.2, 51.5, 46.7, 45.0, 40.4, 37.9, 37.5, 37.2, 37.0,
35.2, 34.9, 31.4, 28.9, 27.8, 26.3, 25.7, 24.2, 22.5, 21.5, 21.2,
18.7, 16.4, 14.9, 14.8, 14.1, 13.1
ESI-MS (m/z): 641.5 [M+Na].sup.+
The method for preparing its sodium salt was similar to that in
Example 5.
Example 7
Preparation of 3-O-(.alpha.-L-arabopyranosyl) ursolic
acid-28-O-(.beta.-D-glucopyranosyl) ester
Similar to the method in Example 4, the intermediate product,
ursolic acid-28-O-(.beta.-D-glucopyranosyl) ester, was obtained
from ursolic acid as a starting material. By using
2,3,4-tri-O-benzoyl-.beta.-L-arabopyranosyl-trichloroacetimidate as
a secondary glycosyl donor under a similar condition of
glycosylation and using NaOMe for deprotection,
3-O-(.alpha.-L-arabopyranosyl) ursolic
acid-28-O-(.beta.-D-glucopyranosyl) ester was obtained.
.sup.1HNMR (600 MHz, CD.sub.3OD): .delta.ppm 5.36 (d, J=8.2 Hz, 1H,
H-1''), 5.26 (t, 1H, H-12), 4.29 (d, J=6.7 Hz, 1H, H-1'), 3.80-3.86
(m, 3H), 3.69 (dd, J=12, 4.6 Hz, 1H, H-6'-1), 3.57-3.58 (m, 1H,
H-6'-2), 3.32-3.43 (m, 4H), 3.15 (dd, J=11.6, 2.4 Hz, 1H), 2.24 (d,
J=11.2 Hz, 1H, H-18), 1.13 (3H, s), 1.06 (3H, s), 0.99 (6H, s),
0.91 (d, J=6.3 Hz, 3H), 0.86 (3H, s), 0.85 (3H, s), 0.79 (d, J=11.7
Hz, 1H, H-5)
.sup.13CNMR (150 MHz, CD.sub.3OD): .delta.ppm 176.5 (C-28), 137.7
(C-13), 125.9 (C-12), 105.7 (C-1'), 94.3 (C-1''), 89.3 (C-3), 77.2,
76.9, 72.9, 72.5, 71.4, 69.8, 68.1, 64.9, 61.1, 55.6, 52.8, 48.2,
41.9, 39.6, 39.0, 38.9, 38.8, 38.6, 36.5, 36.1, 32.9, 30.3, 29.3,
27.9, 27.2, 25.7, 23.9, 23.0, 22.6, 20.1, 17.9, 16.5, 16.2, 15.6,
14.7
ESI-MS (m/z): 773.4 [M+Na]+.sub.o
The compound in this example is the closest to
3-O-(.alpha.-L-arabopyranosyl)-19-hydroxy-ursolic
acid-28-O-(.beta.-D-glucopyranosyl) ester with the highest activity
isolated from sanguisorba, and only lacks a 19-hydroxy. The
compound of this example can be conveniently synthesized using
ursolic acid as a starting material by the method described above,
which is easy to put into industrial practice. However, the
synthesis of said compound from sanguisorba is a problem, since the
raw material, 19-hydroxy-ursolic acid, is not easy to get and,
moreover, it is difficult to synthesize it starting from ursolic
acid. In addition, there are certain limitations in separating the
compound in large quantities directly from natural products. Thus,
its use in the medicinal field is, in a way, restricted. One of the
creative aspects of the present invention lies in providing a
series of compounds which are easier to obtain, can be synthesized
starting from the cheap and accessible ursolic acid and oleanolic
acid, and have an enhanced activity, thus they can be applied
widely to the medicinal field.
Example 8
Preparation of 3-O-(.alpha.-L-arabopyranosyl) ursolic
acid-28-O-(.beta.-cellobiosyl) ester
It was obtained by a sililar method.
[.alpha.].sub.D.sup.20=+30.8 (C0.9, MeOH)
.sup.1HNMR (600 MHz, CD3OD): .delta.ppm 5.36 (1H, d, J=8.3 Hz),
5.24 (1H, t), 4.43 (1H, d, J=7.9 Hz), 4.28 (1H, d, J=6.7 Hz),
3.21-3.89 (16H, m), 3.13 (1H, dd, J=11.4, 4.1 Hz), 2.22 (1H, d,
J=11.4 Hz), 1.11 (3H, s), 1.04 (3H, s), 0.96 (6H, s), 0.89 (3H, d,
J=6.4 Hz), 0.84 (3H, s), 0.83 (3H, d, J=6.9 Hz), 0.78 (1H, d,
J=11.2 Hz)
ESI-MS (m/z): 912 [M].sup.+
Example 9
Preparation of 3-O-(.alpha.-L-rhamnopyranosyl) ursolic acid
Similar to the method in Example
5,2,3,4-tri-O-benzoyl-.beta.-L-arabopyranosyl-trichloroacetimidate
was used as a glycosyl donor. After glycosylation followed by
deprotection, 3-O-(.alpha.-L-rhamnopyranosyl) ursolic acid was
obtained. [.alpha.].sub.D.sup.20=+5.7 (C0.74, MeOH)
.sup.1HNMR (600 MHz, d.sub.5-pyridine): .delta.ppm 5.48 (1H, m),
5.32 (1H, s), 4.55 (1H, t, J=1.6 Hz), 4.46 (1H, dd, J=10.4, 3.4
Hz), 4.26-4.34 (2H, m), 3.17 (1H, dd, J=11.8, 4.4 Hz), 2.62 (1H, d,
J=11.4 Hz), 2.31 (1H, dt, J=13.4, 4.4 Hz), 2.12 (1H, dt, J=4.0,
12.6 Hz), 1.87-2.15 (6H, m), 1.66 (3H, d, J=5.8 Hz), 1.56 (1H, m),
1.24 (s, 3H), 1.02 (3H, s), 1.00 (3H, d, J=6.4 Hz), 0.96 (3H, d,
J=6.4 Hz), 0.93 (3H, s), 0.83 (3H, s), 0.79 (3H, s), 0.72 (1H, d,
J=11.6 Hz)
.sup.13CNMR (150 MHz, d.sub.5-pyridine): .delta.ppm 180.2, 138.2,
125.5, 103.0, 89.0, 72.7, 71.2, 71.1, 68.5, 55.3, 53.0, 48.2, 48.0,
47.2, 41.9, 39.4, 39.0, 38.6, 38.4, 36.7, 36.5, 32.9, 30.4, 27.8,
27.4, 25.2, 23.9, 23.0, 22.7, 20.2, 18.0, 16.4 (d), 16.2, 15.6,
14.6
ESI-MS (m/z): 1203 [2M-1].sup.+, 601 [M-1]+
Example 10
Preparation of 3-O-(.beta.-D-galactopyranosyl) ursolic acid
Similar to the method in Example
5,2,3,4,6-tetra-O-benzoyl-.alpha.-D-galactopyranosyl-trichloroacetimidate
was used as a glycosyl donor. After glycosylation followed by
deprotection, 3-O-(.beta.-D-galactopyranosyl) ursolic acid was
obtained. [.alpha.].sub.D.sup.20=+13.8 (C0.8, MeOH)
.sup.1HNMR (600 MHz, d5-pyridine): .delta.ppm 5.48 (1H, t, J=3.2
Hz), 4.93 (1H, d, J=6.6 Hz), 4.57 (1H, d, J=3.2 Hz), 4.43-4.48 (3H,
m), 4.16 (1H, dd, J=9.6, 3.4 Hz), 4.11 (1H, t, J=6.2 Hz), 3.41 (1H,
dd, J=11.8, 4.4 Hz), 2.62 (1H, d, J=11.6 Hz), 2.20-2.32 (2H, m),
2.12 (1H, m), 1.57 (1H, t, J=8.0 Hz), 1.31 (3H, s), 1.26 (3H, s),
1.01 (3H, s), 1.00 (3H, d, J=5.8 Hz), 0.96 (3H, d, J=5.2 Hz), 0.96
(3H, s), 0.85 (3H, s), 0.80 (1H, d, J=11.8 Hz)
ESI-MS (m/z): 641 [M+Na].sup.+
Example 11
Preparation of 3-O-(.alpha.-L-arabopyranosyl) oleanolic
acid-28-O-(.alpha.-L-arabopyranosyl) ester
Similar to the method in Example
1,2,3,4-tri-O-benzoyl-.beta.-L-arabopyranosyl-trichloroacetimidate
was used as a glycosyl donor. After a glycosylation reaction
followed by deprotection using sodium methoxide, the title compound
was obtained.
[.alpha.].sub.D.sup.20=+35.5 (C0.71, MeOH)
.sup.1HNMR (600 MHz, CD.sub.3OD): .delta.ppm 5.41 (1H, d, J=5.7 Hz,
H-1''), 5.27 (1H, t, H-12), 4.27 (1H, d, J=6.7 Hz), 3.79-3.90 (4H,
m), 3.66-3.70 (2H, m), 3.55-3.58 (2H, m), 3.49-3.52 (2H, m), 3.13
(1H, dd, H-3), 2.00-2.06 (1H, m, H-18), 1.15, 1.04, 0.95, 0.94,
0.90, 0.83, 0.79 (each 3H, s, Me.times.7)
ESI-MS (m/z): 743.5 [M+Na].sup.30
Example 12
Preparation of 3-O-(.beta.-D-glucopyranosyl) oleanolic
acid-28-O-(.beta.-D-glucopyranosyl) ester
2,3,4,6-tetra-O-benzoyl-.alpha.-D-glucopyranosyl-trichloroacetimidate
was used as a glycosyl donor. Similar to Example 1, the title
compound was obtained.
.sup.1HNMR (600 MHz, CD.sub.3OD): .delta.ppm 5.37 (d, J=8.1 Hz, 1H,
H-1''), 5.25 (m, 1H, H-12), 4.31 (d, J=7.86 Hz, 1H, H-1'),
3.17-3.84 (13H, m), 2.85 (1H, m), 1.16, 1.05, 0.95, 0.93, 0.89,
0.84, 0.80 (s each, 3H each, Me.times.7)
ESI-MS (m/z): 803 [M+Na].sup.+
Example 13
Preparation of 3-O-(.beta.-D-glucopyranosyl) oleanolic acid
Similar to Example 5.
.sup.1H NMR (600 MHz, CD.sub.3OD): .delta.ppm 5.26 (1H, t, J=3.6
Hz), 4.34 (1H, d, J=7.9 Hz), 3.86 (1H, dd, J=11.8, 2.3 Hz), 3.68
(1H, dd, J=11.8, 5.3 Hz), 3.19-3.37 (5H, m), 2.88 (1H, m), 1.18,
1.08, 0.98, 0.97, 0.92, 0.88, 0.86 (each 3H, s), 0.81 (1H, d,
J=11.6 Hz)
.sup.13C NMR (150 MHz, CD.sub.3OD): .delta.ppm 181.5 (C-28), 144.1
(C13), 122.0 (C12), 105.3 (C'1), 89.4 (C3), 76.9-70.3 (C'4, C'5,
C'3, C'2), 61.4 (C6), 55.7 (C5), 48.2 (C9), 47.2 (C19), 46.5 (C17),
46.1 (C18), 41.5 (C14), 39.2 (C8), 38.8 (C1), 38.4 (C4), 36.5
(C10), 33.7 (C21), 32.7 (C29), 32.6 (C7), 32.2 (C22), 30.4 (C20),
27.5 (C23), 27.2 (C15), 25.6, 25.0 (C27, C25), 23.1 (C-30), 22.8
(C11), 22.7 (C16), 18.0 (C6), 16.4 (C26), 15.6 (C24), 14.5
(C25)
The method for preparing its sodium salt was the same as
before.
Example 14
Preparation of 3-O-(.beta.-D-glucopyranosyl) oleanolic acid
Similar to Example 5.
.sup.1H NMR (600 MHz, d.sub.6-DMSO): .delta.ppm 12.00 (brs, 1H),
5.16 (s, 1H, H12), 4.71 (d, J=4.7 Hz, 1H), 4.60 (d, J=5.5 Hz, 1H),
4.50 (t, J=5.8 Hz, 1H), 4.29 (d, J=4.4 Hz, 1H), 4.10 (d, J=7.3 Hz,
1H), 3.6 (t, J=3.7 Hz, 1H, H4), 3.54-3.50 (m, 1H), 3.39-3.42 (m,
1H), 3.23-3.29 (m, 3H), 3.02 (dd, J=4.7, 11.4 Hz, 1H, H3), 2.80
(dd, J=3.3, 13.6 Hz, 1H, H18), 1.89-1.93 (m, 1H), 1.80-1.77 (m,
3H), 1.09 (s, 3H), 0.98 (S, 3H), 0.87 (S, 9H), 0.75 (s, 3H), 0.71
(s, 3H)
.sup.13CNMR (150 MHz, d6-DMSO): .delta.ppm 178.6 (C28), 143.8
(C13), 121.5 (C12), 106.0 (C'1), 87.8 (C3), 74.8-68.0 (C'4, C'5,
C'2, C'3), 60.3 (C'6), 54.9 (C5), 47.0 (C9), 45.7 (C19), 45.2
(C17), 41.2 (C18), 40.8 (C14), 38.8 (C8), 38.7 (C1), 38.1 (C4),
36.3 (C10), 33.3 (C21), 32.8 (C29), 32.3 (C7), 32.1 (C22), 30.4
(C20), 27.6 (C23), 27.1 (C15), 25.6 (C27), 25.5 (C25), 23.3 (C30),
22.9 (C11), 22.6 (C16), 17.7 (C6), 16.8 (C26), 16.4 (C24), 15.1
(C25)
Example 15
Preparation of 3-O-(.beta.-D-xylopyranosyl) oleanolic acid
2,3,4-tri-O-benzoyl-.alpha.-D-xylopyranosyl-trichloroacetimidate
was used as a glycosyl donor. Similar to Example 5,
3-O-(.beta.-D-xylopyranosyl) oleanolic acid was obtained.
.sup.1H NMR (600 MHz, d.sub.6-DMSO): .delta.ppm 12.00 (brs, 1H,
COOH), 5.16 (brs, 1H, H12), 4.89-4.91 (m, 3H), 4.11 (d, J=7.8 Hz,
1H, H1), 3.63 (dd, J=5.0, 11.0 Hz, 1H), 3.21-3.26 (m, 1H),
2.92-3.07 (m, 4H), 2.73 (dd, J=3.7, 13.7 Hz, 1H), 1.89-1.93 (m,
1H), 1.79 (brd, J=8.7 Hz, 2H), 1.09 (s, 3H), 0.97 (s, 3H), 0.87 (s,
6H), 0.85 (s, 3H), 0.75 (s, 3H), 0.71 (s, 3H)
.sup.13CNMR (150 MHz, d.sub.6-DMSO): .delta.ppm 178.6 (C28), 143.8
(C13), 121.5 (C12), 106.2 (C'1), 87.6 (C3), 76.7 (C'3), 73.7 (C'2),
69.6 (C'4), 65.5 (C'5), 54.9 (C5), 46.9 (C9), 45.7 (C19), 45.4
(C17), 41.3 (C18), 40.8 (C14), 38.8 (C8), 38.7 (C1), 37.9 (C4),
36.3 (C10), 33.3 (C21), 32.8 (C29), 32.3 (C7), 32.1 (C22), 30.3
(C20), 27.4 (C23), 27.2 (C15), 25.7 (C27), 25.5 (C2), 23.4 (C30),
22.9 (C11), 22.6 (C16), 17.7 (C6), 16.8 (C26), 16.4 (C24), 15.0
(C25)
Example 16
Preparation of 3-O-(.alpha.-L-arabopyranosyl) oleanolic acid
Similar to Example 5.
.sup.1H NMR (600 MHz, d.sub.6-DMSO): .delta.ppm 12.01 (s, 1H,
COOH), 5.15 (t, J=3.6 Hz, 1H, H12), 4.81 (d, J=4.7 Hz, 1H), 4.52
(d, J=5.9 Hz, 1H), 4.47 (d, J=4.4 Hz, 1H), 4.11 (d, J=6.2 Hz, 1H,
H1), 3.64 (dd, J=3.3, 12.5 Hz, 1H), 3.58 (brs, 1H), 3.33-3.34 (m,
3H), 3.00 (dd, J=4.4, 11.4 Hz, 1H, H3), 2.74 (dd, J=5.4, 13.6 Hz,
1H), 1.88-1.93 (m, 1H), 1.79 (dd, J=3.3, 8.4 Hz, 2H, H11), 1.09 (s,
3H, H-27), 0.96 (s, 3H, H23), 0.87 (s, 9H), 0.75 (s, 3H), 0.71 (s,
3H)
.sup.13CNMR (150 MHz, d.sub.6-DMSO): .delta.ppm 178.6 (C28), 143.8
(C13), 121.5 (C12), 105.8 (C1), 87.6 (C3), 72.7, 70.9, 67.6, 65.1,
54.9, 46.9, 45.7, 45.4, 41.3, 40.8, 38.8, 38.7, 38.0, 36.3, 33.3,
32.8, 32.3, 32.0, 30.4, 27.6, 27.2, 25.7, 25.5, 23.4, 22.9, 22.6,
17.7, 16.8, 16.4, 15.1
The method for preparing its sodium salt was the same as before.
The spectral data of the compounds in Examples 13, 14, 15 and 16
are identical to the data in this literature, Journal of Ocean
University of China (2005, 35(4): 635-640).
Example 17
Preparation of oleanolic acid-28-O-(.beta.-D-glucopyranosyl)
ester
2,3,4,6-tetra-O-benzoyl-.alpha.-D-glucopyranosyl-bromide and
oleanolic acid were used as raw materials. After phase-transfer
glycosylation similar to Example 4 followed by deprotection using
sodium methylate, the title compound was obtained.
m.p. 180-183.degree. C., [.alpha.].sub.D.sup.20=+22 (C0.2,
pyridine)
IR(KBr) cm.sup.-1: 3435, 2933, 2862, 1695, 1461, 1386, 1181, 1030,
996, 762
.sup.1HNMR (600 MHz, d.sub.5-pyridine): .delta.ppm 6.19 (1H, d, J=8
Hz, H'-1), 5.35 (1H, brs, H12), 1.18, 1.17, 1.13, 1.06, 0.91, 0.90,
0.86 (21H, s, 7 Me)
.sup.13CNMR: .delta.ppm 176.9, 144.7, 123.4, 92.3, 79.8, 79.4,
78.6, 74.6, 71.6, 62.7, 56.3, 48.7, 47.5, 46.7, 42.3, 40.4, 40.0,
39.5, 37.9, 34.5, 33.6, 33.0, 31.3, 30.5, 29.3, 28.8, 28.6, 26.6,
24.3, 23.9, 24.3, 24.2, 23.9, 18.0, 17.0, 16.1
ESI-MS (m/z): 641 [M+Na].sup.30
Example 18
Preparation of oleanolic acid-28-O-(.beta.-gentiobiosyl) ester
Fully acetylated gentiobiosyl-bromide and oleanolic acid were used
as raw matarials. After phase-transfer glycosylation similar to
Example 4, followed by deprotection using sodium methylate, the
title compound was obtained. m.p. 248-250.degree. C.
.sup.1H NMR (600 MHz, CD.sub.3OD): .delta.ppm 5.41 (1H, d, J=7.8
Hz, H'1), 5.32 (1H, d, H12), 4.41 (1H, d, J=8.7 Hz, H''-1), 4.18
(1H, d, J=11.6 Hz), 3.91 (1H, d, J=13.6 Hz), 3.83 (1H, dd, J=6, 5.5
Hz), 3.73 (1H, dd, J=5.5, 5.5 Hz), 1.22, 1.03, 1.01, 1.00, 0.97,
0.87, 0.84 (21H, s, 7.times.Me)
Example 19
Preparation of 3-O-(.beta.-D-glucuronopyranosyl) oleanolic acid
Under reaction conditions similar to those in Example 5, the title
compound was obtained by glycosylation and the subsequent
deprotection.
.sup.1HNMR (600 MHz, d.sub.5-pyridine): .delta.ppm 5.65 (1H, brs,
H12), 4.82 (1H, d, J=7 Hz, H'-1), 1.33, 1.32, 1.29, 1.04, 0.99,
0.98, 0.79 (21H, s, 7.times.Me)
ESI-MS (m/z): 655 [M+Na].sup.30
Next, the beneficial effects of the present invention will be
confirmed through pharmacodynamic tests.
In vitro experiments of growth rate of bone marrow monocyte
proliferation were carried out on the compounds in the examples,
and in vivo experiments of increasing leucocytes and/or platelets
in mice were carried out on some compounds in the examples. By
comparison of the therapeutic effects of
3-O-(.alpha.-L-arabopyranosyl)-19-hydroxy-ursolic
acid-28-O-(.beta.-D-glucopyranosyl) eater which is a monomer
saponin isolated from sanguisorba, ursolic acid and oleanolic acid,
the result confirms the beneficial effects of the present
invention.
Experimental Example 1
Effect in Promoting Monocytic Proliferation in Bone Marrow of Mice
Cultured In Vitro
15-20 KunMing mice (purchased from Laboratory Animal Center of
Zhengzhou University, Henan) of either sex were killed by cervical
vertebral dislocation. Bilateral thighbones were isolated and caput
femoris was cut off. The marrow was washed repeatedly with
serum-free RPMI1640 (Gibco Corp.), the wash solution containing
marrow cells was collected, sucked and blew repeatedly with a
pipette to make the cells disperse. After standing for a moment,
the supernatant was sucked up and centrifugated at 100 g for 3 min
to collect the cell pellet. The cells were resuspended in a culture
media and brought to a volume of 5 ml. The cell suspension was
added to the upper layer of a centrifugal tube containing 5 ml of a
lymphocyte separation medium of mice and centrifugated at 400 g for
30 min. The milk-white bone marrow mononuclear cell (BMMNC) layer
in the middle of the interface was collected and washed with
serum-free medium RPMI1640 for three times to obtain a BMMNC
suspension, which was inoculated into a 96-well plate at forty
thousand per well after counting. Positive drugs rhG-CSF
(recombinant granulocyte colony-stimulating factor, manufactured by
QI LU Pharmaceutical Co., LTD., Shandong) or test samples of
different concentrations were added, and meanwhile a blank control
group without drug was established. They were cultured in a 5%
CO.sub.2 incubator at 37 for 5 days. Cell proliferation was
measured by a conventional SRB (Sigma) method in which OD at 490 nm
was measured after being dyed with SRB. The growth rates of cell
proliferation after drug treatment were calculated (Growth rate of
cell proliferation=experimental group-blank control group/blank
control group*100%). The experimental results are shown in Table
1.
TABLE-US-00001 TABLE 1 Sample number and growth rate of monocytic
proliferation in bone marrow of mice (%) Growth Rate of
Proliferation Sample %, Number Compound 1 .mu.g/ml Blank 0 G-1
3-O-(.alpha.-L-arabopyranosyl) ursolic acid-28-O- 31.3
(.alpha.-L-arabopyranosyl) ester G-2
3-O-(.alpha.-L-rhamnopyranosyl) ursolic acid-28-O- 19.9
(.alpha.-L-rhamnopyranosyl) ester G-3 3-O-(.beta.-D-glucopyranosyl)
ursolic acid-28-O- 25.2 (.beta.-D-glucopyranosyl) ester G-4
3-O-(.alpha.-L-arabopyranosyl) ursolic acid-28-O-(.beta.- 18.0
cellobiosyl) ester G-5 ursolic acid-28-O-(.alpha.-L-arabopyranosyl)
ester 20.1 G-6 3-O-(.alpha.-L-arabopyranosyl) ursolic acid 29.9 G-7
3-O-(.beta.-D-glucopyranosyl) ursolic acid 23.7 G-8
3-O-(.alpha.-L-arabopyranosyl) ursolic acid-28-O- 29.5
(.beta.-D-glucopyranosyl) ester G-9 3-O-(.alpha.-L-rhamnopyranosyl)
ursolic acid 23.4 G-10 3-O-(.beta.-D-galactopyranosyl) ursolic acid
24.2 G-11 3-O-(.alpha.-L-arabopyranosyl) oleanolic acid-28-O- 26.8
(.alpha.-L-arabopyranosyl) ester G-12 3-O-(.beta.-D-glucopyranosyl)
oleanolic acid-28-O- 25.4 (.beta.-D-glucopyranosyl) ester G-13
3-O-(.beta.-D-glucopyranosyl) oleanolic acid 21.6 G-14
3-O-(.beta.-D-galactopyranosyl) oleanolic acid 19.9 G-15
3-O-(.beta.-D-xylopyranosyl) oleanolic acid 20.6 G-16
3-O-(.alpha.-L-arabopyranosyl) oleanolic acid 21.6 G-17 oleanolic
acid-28-O-(.beta.-D-glucopyranosyl) ester 20.6 G-18 oleanolic
acid-28-O-(.beta.-gentiobiosyl) ester 19.0 G-19
3-O-(.beta.-D-glucuronopyranosyl) oleanolic acid 20.9 G-20
oleanolic acid 4.5 G-21 ursolic acid 7.9 G-22
3-O-(.alpha.-L-arabopyranosyl)-19-hydroxy-ursolic 15.2
acid-28-O-(.beta.-D-glucopyranosyl) ester Positive G-CSF 33.2 (5
pg/ml)
Results and Discussion
As shown in Table 1, the growth rates of proliferation of samples
G1-G19 according to the present invention are more than 10% higher
than those of ursolic acid and oleanolic acid, and the difference
is remarkable. G-22 isolated from sanguisorba is a compound with
the strongest activity among those disclosed in Chinese Patent No.
CN03135776. Sample G-8 has a structure quite similar to that of
G-22 and is only different in lacking 19-hydroxy. Results shown in
the above table suggest that the saponin with ursolic acid or
oleanolic acid as an aglycon according to the present invention
have advantages of simple synthesis and enhanced activity, compared
with the saponin with pomolic acid as an aglycon.
It also can be seen from the above data that with respect to G-1,
G-3, G-6 and G-8 of ursolic acid saponin, G-11 and G-12 of
oleanolic acid saponin have higher growth rates of proliferation,
so the above-mentioned compounds were preferably selected and
studied using an in vivo experimental method.
Further, in vivo experiments were carried out on the compounds
involved in the present invention using animal models of
cyclophosphamide-induced leukopenia in mice. Experimental methods
are briefed as follows: Healthy Kunming mice, of which half were
male and the other half were female, were divided into groups, each
group incuding 18 mice, and those mice weighing 18-22 g were
selected and divided into groups randomly according to their body
weights. Each group was given a different drug, wherein the
negative control group and model control group were given
physiological saline of the same volume, and the positive control
group was treated with granulocyte colony-stimulating factors 40
.mu.g/kg through subcutaneous injection (injection volume: 0.1
ml/10 g). After administration for 3 days, except the negative
control group, other treatment groups were treated with 100 mg/kg
cyclophosphamide (CY) through lumbar injection once a day for three
days to cause a decrease of leukocytes in mice. A drop of blood (20
.mu.l) was taken from posterior orbital venous plexus to detect the
hemogram to observe the situation of leukocytes and platelets on
day 1, day 3 and/or day 5 after the final lumbar injection of
cyclophosphamide (i.e day 1, day 3 and/or day 5 after modeling)
(Note: Each treatment group was administrated incessantly). The
mice were killed on day 5 after the final lumbar injection of
cyclophosphamide (i.e day 5 after modeling), and the leukocytes
(WBC) and platelets (PLT) of the hemogram index were counted.
Experimental Example 2
Effect of Ursolic Acid Saponin on Leukocytes (WBC) of Animal Model
of Cyclophosphamide-Induced Leukopenia in Mice
This experiment mainly studies the effect of G-1, G-6 and G-8 of
ursolic acid saponin in increasing leukocytes, and compares the
therapeutic effects of aglycon of ursolic acid and oleanolic acid
and of the compound G-22 which has been disclosed by a patent. The
results are shown in Table 2:
TABLE-US-00002 TABLE 2 Effect of ursolic acid saponin on leukocytes
(WBC) of animal model of CY-induced leukopenia in mice Day1 after
Day 3 after modeling modeling Day 5 after Group Dosage Statistics
Statistics modeling negative control -- 8.89 .+-.
3.45**.star-solid..star-solid. 9.28 .+-.
3.44**.star-solid..star-solid. 9.14 .+-. 4.9 group model control --
0.87 .+-. 0.27 3.74 .+-. 1.92 8.17 .+-. 3.6 group G-1 0.8 mg/kg
1.14 .+-. 0.56.DELTA. 7.85 .+-. 3.00**.star-solid..star-solid.
15.46 .+-. 10.44* G-6 0.8 mg/kg 1.19 .+-. 0.47* 7.13 .+-.
2.61**.star-solid. 17.16 .+-. 14.13* G-8 0.8 mg/kg 0.85 .+-. 0.39
6.91 .+-. 2.54**.star-solid. 15.32 .+-. 9.07** G-20 0.8 mg/kg 0.99
.+-. 0.40 3.88 .+-. 2.01 7.00 .+-. 3.22 G-21 0.8 mg/kg 1.01 .+-.
0.47 3.94 .+-. 1.73 7.07 .+-. 3.14 G-22 0.8 mg/kg 1.08 .+-.
0.34.DELTA. 5.17 .+-. 1.77* 11.87 .+-. 9.53 G-CSF 40 .mu.g/kg 1.17
.+-. 0.33* 5.93 .+-. 3.10* 13.18 .+-. 5.53** Note: Compared with
the model control group between groups, *P < 0.05, **P <
0.01, .DELTA.stands for a good trend. Compared with G-22 between
groups, .star-solid.P < 0.05, .star-solid..star-solid.P <
0.01.
It can be seen from the above table that there is an obvious
difference in increasing leukocytes under the dose of 0.8 mg/kg
between G-1, G-6 and G-8 of ursolic acid saponin provided in the
present invention and the disclosed G-22, especially on day 3 after
modeling. However, G-20 and G-21 of aglycon have no obvious effect
in increasing leukocytes.
Experimental Example 3
Effect of Ursolic Acid Saponin and Oleanolic Acid Saponin on
Leukocytes (WBC) and Platelets of Animal Model of
Cyclophosphamide-Induced Leukopenia in Mice
The purpose of this experiment is to study the effect of G-1, G-3,
G-11 and G-12 of ursolic acid saponin and oleanolic acid saponin in
increasing platelets, and to observe whether G-11 and G-12 of
oleanolic acid saponin have an effect in increasing leukocytes. The
experimental method is the same as above, and the results are shown
in the following table.
TABLE-US-00003 TABLE 3 Effect of ursolic acid saponin and oleanolic
acid saponin on leukocytes of animal model of CY-induced leukopenia
in mice Day 5 after Dosage Day 1 after modeling modeling Group
(/kg) Statistics Statistics negative control -- 6.68 .+-.
2.27**.star-solid..star-solid. 6.52 .+-. 2.24 group model control
group -- 0.50 .+-. 0.19 5.42 .+-. 1.99 G-1 0.8 mg/kg 0.98 .+-.
0.55** 9.33 .+-. 5.17** G-3 0.8 mg/kg 1.16 .+-. 0.53** 5.62 .+-.
2.78 G-11 0.8 mg/kg 0.94 .+-. 0.77** 7.55 .+-. 4.28 G-12 0.8 mg/kg
1.01 .+-. 0.55** 5.48 .+-. 4.39 G-22 0.8 mg/kg 0.92 .+-. 0.28**
8.81 .+-. 6.44* G-CSF 40 .mu.g/kg 0.63 .+-. 0.20* 9.70 .+-. 5.28**
Note: Compared with the model control group between groups, *P <
0.05, **P < 0.01;
TABLE-US-00004 TABLE 4 Effect of ursolic acid saponin and oleanolic
acid saponin on platelets of animal model of CY-induced leukopenia
in mice Day 1 after Dosage modelng Day 5 after modeling Group (/kg)
Statistics Statistics negative -- 742.30 .+-.
199.09**.star-solid..star-solid. 678.30 .+-.
178.03**.star-solid..star-solid. control group model -- 388.14 .+-.
116.19 176.50 .+-. 115.24 control group G-1 0.8 mg/kg 504.67 .+-.
117.81** 413.00 .+-. 218.95*.star-solid..star-solid. G-3 0.8 mg/kg
473.12 .+-. 144.68* 341.93 .+-. 186.39**.star-solid. G-11 0.8 mg/kg
465.00 .+-. 99.28* 385.73 .+-. 198.30**.star-solid. G-12 0.8 mg/kg
456.88 .+-. 77.20* 390.86 .+-. 203.71**.star-solid..star-solid.
G-22 0.8 mg/kg 491.36 .+-. 126.28* 210.00 .+-. 119.38 G-CSF 40
.mu.g/kg 408.38 .+-. 86.38 206.64 .+-. 140.14 Note: Compared with
the model control group between groups, *P < 0.05, **P <
0.01, .DELTA. stands for a good trend. Compared with G-22 between
groups, .star-solid.P < 0.05,
.star-solid..star-solid.<0.01.
As can be seen from Table 3 and Table 4 showing the results of
Experiment 3, G-1 of ursolic acid saponin has an obvious effect in
increasing leukocytes and platelets. There is a significant
difference in increasing leukocytes and platelets between G-1, G-3
of ursolic acid saponin and G-22 on day 5 after modeling. G-11 and
G-12 of oleanolic acid saponin have an obvious effect in increasing
leukocytes at day 1 after modeling, thereby having a certain effect
in increasing leukocytes. The effect in increasing platelets is
obvious on day 1 and day 5, and there is a significant difference
on day 5 compared with G-22. The positive drug G-CSF has no obvious
effect in increasing platelets.
Experimental Example 4
In order to illustrate the present invention in detail, with a
model induced by .sup.60Co-.gamma. rays, the effects of G-1 and
G-22 on leukocytes of chemotherapy-induced-leukopenia mice were
compared. The specific operation and the results are as follows.
The dosage regimen is shown in the following table.
TABLE-US-00005 TABLE 5 Drug dosage regimen Drug Drug Drug Test
Concentration Volume Dosage Administration Administration Animal
Group Material (mg/ml) (ml/kg) (mg/kg) Route Time(day) Mice normal
0.9% / 20 / i.g 21 control physiological group saline model 0.9% /
20 / i.g 21 group physiological saline G-1 G-1 0.04 20 0.80 i.g 21
G-22 G-22 0.04 20 0.80 i.g 21 positive G-CSF 4 .mu.g/ml 10 40
.mu.g/kg s.c day 5 after (G-CSF) irradiation
Experimental Methods and Results:
Healthy Kunming mice, of which half were male and the other half
were female, were divided into 6 groups randomly, with 17-18 in
each group. Blood was taken from caudal vein to determine the
normal hemogram (leucocyte, erythrocyte, platelet and hemoglobin),
and adjustments were made according to body weights and hemogram
results. The drugs were adminstrated at a dosage ig as shown in
Table 5. The normal control group and model group were given
isometric physiological saline once a day for 7 days, and then the
post-radiation hemogram was determined. Except the normal control
group, other groups were each subjected to a whole body exposure to
.sup.60Co-.gamma. rays at a total dose of 3.0 Gy to prepare the
models of leukopenia in mice, followed by further administration
for 14 days. The G-CSF group was adminitrated through subcutaneous
injection for 5 days from the irradiation day on and the hemogram
was determined by taking blood respectively on day 3, day 7 and day
14 after irradiation. The specific results are shown in the
following table:
TABLE-US-00006 TABLE 6 The effect of test drug on WBC of mice model
of.sup.60Co-.gamma.-induced leukopenia (109/L) Detection Time Point
Dosage 1 week after Day 3 after Day 7 after Day 14 after Group
(mg/kg) Number Normal administration irradiation irradiation
irradia- tion Normal / 17 8.51 .+-. 1.88 10.08 .+-. 2.89 10.71 .+-.
3.63 12.30 .+-. 3.67 11.35 .+-. 3.92 Group Model / 18 8.47 .+-.
2.37 10.03 .+-. 2.32 2.48 .+-. 0.97.sup..DELTA..DELTA. 3.45 .+-.
1.13.sup..DELTA..DELTA. 5.06 .+-. 1.08.sup..DELTA..DELTA. Group G-1
0.80 17 8.96 .+-. 1.58 9.21 .+-. 2.16 2.06 .+-. 0.75 4.59 .+-.
1.52** 6.37 .+-. 1.54** G-22 0.80 17 8.56 .+-. 2.43 9.50 .+-. 3.17
2.21 .+-. 0.92 4.08 .+-. 1.50 5.44 .+-. 1.13 G-CSF 40 .mu.g 18 8.64
.+-. 2.01 9.89 .+-. 3.30 3.71 .+-. 1.11** 4.56 .+-. 1.17** 7.01
.+-. 1.52** (Compared with normal group, .sup..DELTA..DELTA.P <
0.01; .sup..DELTA.P < 0.05; Compared with model group, **P <
0.01, *P < 0.05.)
Table 6 shows: significantly different from the normal group, the
peripheral blood WBC of model mice was decreased obviously on day
3, day 7 and day 14 after irradiation; and after gastric perfusion
in the G-1 group, the leucocyte count of the leukopenia mice was
increased obviously, and there is a significant difference compared
to the model group on day 7 and day 14 after irradiation, whereas
G-22 has no evident effect.
Experimental Example 5
Comparison of Hemolytic Activities
The test samples were dissolved in DMSO/physiological saline (1:4)
as a solvent to generate the tested concentration gradients of 40,
80, 120, 160, 200 .mu.g/ml. Blood (25 ml) was taken from a rabbit
heart, poured into an erlenmeyer flask containing glass beads and
shook gently for 10 min to remove fibrinogen so as to convert the
blood into defidrinated blood. The resultant blood was transferred
into a graduation centrifugal tube. About 10 times physiological
saline was added, shook up and centrifuged at 2000 r/min for 5
minutes. The supernatant was removed, and the precipitated
erythrocytes were washed with physiological saline again by the
above method for 4 times until the supernatant was colorless and
transparent. The resulant erythrocytes were prepared into a 10%
suspension using physiological saline for testing. 250 .mu.l of the
10% erythrocyte suspension was added into 2.5 ml of a tested
sample, solvent (blank control) or distilled water (positive
control), mixed thoroughly, and immediately placed in a thermotank
maintained at 37.degree. C. for incubation; and it was taken out 1
hour later and centrifuged at 3000 r/min for 5 minutes. The
supernatant was taken for determining the OD value at 540 nm with a
spectrophotometer. Hemolysis (Hemolysis Percentage)=(absorbance of
sample-absorbance of blank control).times.100%/(absorbance of
positive control-absorbance of blank control); and the data
processing was performed by SPSS13.0 statistical software with drug
concentration as an independent variable X and hemolysis as a
dependent variable Y to obtain a linear regression equation; then,
the drug concentration causing 50% hemolysis, i.e HD.sub.50, was
calculated according to the equation. A P-value <0.05 was
considered statistically significant.
TABLE-US-00007 TABLE 7 Regression equation and HD.sub.50 of G
series samples Regression De- Equation Samole termination
(y-hemolysis, x-drug HD.sub.50 No. Coefficient F P concentration)
(.mu.g/ml) G-1 0.822 13.844 0.034 y = 9.290 + 0.118x 345 G-3 0.928
38.856 0.008 y = -30.861 + 0.586x 137.99 G-6 0.857 17.939 0.024 y =
-28.305 + 0.645x 121.40 G-8 0.889 23.961 0.016 y = -12.054 + 0.887x
69.96
It can be seen from the above table that G-1 has the highest
HD.sub.50, showing that among the several samples, G-1 is the most
difficult to cause hemolysis, thus being preferably selected.
It is demonstrated by the above pharmacological tests that the
compounds according to the present invention not only have an
effect in obviously increasing leucocytes and/or platelets, but
also have a stronger pharmacological acitivity and a significant
difference compared with the pomolic acid saponin isolated from the
traditional Chinese medicine-sanguisorba. The efficacy of
bisglycosyl ursolic acid saponin G1 is particularly optimal. It is
known from the hemolysis test that bisglycosyl ursolic acid saponin
G1 among the compounds of the present invention has the highest
value of HD.sub.50 and a high safety; and based on the results of
tests regarding efficacy and safety, bisglycosyl ursolic acid
saponin G1 is the best choice of the compounds according to the
present invention.
Preparation of a Pharmaceutical Composition of the Present
Invention
Tablet:
TABLE-US-00008 Compostion: Amount (mg/tablet) G-1 5 starch 50
microcrystalline cellulose 40 magnesium stearate 2 sodium
carboxymethylcellulose 5
G-1, starch, microcrystalline cellulose and sodium
carboxymethylcellulose were mixed thoroughly according to the above
ratio, wetted with water, granulated, dried and pelletized, prior
to the addition of magnesium stearate. The mixture was mixed
thoroughly and subjected to tabletting to obtain a tablet.
Injection:
50 mg of G-12 was weighed, dissolved in a proper amount of
anhydrous ethyl alcohol, and added with 1 g of polyoxyethylene
castor oil ester to dissolve it completely. A glucose injection was
added to dilute the solution to 100 ml, and 0.05% activited carbon
for injection was added, then the temperature was kept at
80.degree. C. for 15 minutes. The mixture was filtered by sintered
glass and millipore membrane filters. The filtrate was subpackaged
into 2 ml ampoules. Each preparation unit comprises 1 mg of
G-12.
Capsule:
10 g of G-6 and 400 g of microcrystalline cellulose were mixed
thoroughly and filled into 2,000 No. 1 capsules. Each preparation
unit comprises 5 mg of G-6.
Industrial Applicability
The present invention utilizes the cheap and accessible ursolic
acid and oleanolic acid which are widely present in natural plants
as raw materials, introduces hydrophilic groups, i.e monosaccharyls
or oligosaccharyls, by structural modification, and discovers that
the resulting saponins not only can improve the water solubility of
the parent nucleus, but also have an activity of remarkably
increasing leucocytes and/or platelets, as demonstrated by
pharmacological tests. Compared to pomolic acid saponin isolated
from sanguisorba, they not only have a stronger pharmacological
activity, but also have the advantages of simple synthesis,
adaptation to industrial production, low cost and so on.
* * * * *